Effective gene silencing using type I-E CRISPR system in the multiploid, radiation-resistant bacterium Deinococcus radiodurans.

The extremely radiation-resistant bacterium, Deinococcus radiodurans, is a microbe of importance, both, for studying stress tolerance mechanisms and as a chassis for industrial biotechnology. However, the molecular tools available for use in this organism continue to be limiting, with its multiploid genome presenting an additional challenge. In view of this, the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas tools provide a large repertoire of applications for gene manipulation. We show the utility of the type I-E Cascade system for knocking down gene expression in this organism. A single-vector system was designed for the expression of the Cascade components as well as the crRNA. The type I-E Cascade system was better tolerated than the type II-A dCas9 system in D. radiodurans. An assayable acid phosphatase gene, phoN integrated into the genome of this organism could be knocked down to 10% of its activity using the Cascade system. Cascade-based knockdown of ssb, a gene important for radiation resistance resulted in poor recovery post-irradiation. Targeting the Radiation and Desiccation Response Motif (RDRM), upstream of the ssb, prevented de-repression of its expression upon radiation exposure. In addition to this, multi-locus targeting was demonstrated on the deinococcal genome, by knocking down both phoN and ssb expression simultaneously. The programmable CRISPR interference tool developed in this study will facilitate the study of essential genes, hypothetical genes, and cis-elements involved in radiation response as well as enable metabolic engineering in this organism. Further, the tool can be extended for implementing high-throughput approaches in such studies. IMPORTANCE Deinococcus radiodurans is a microbe that exhibits a very high degree of radiation resistance. In addition, it is also identified as an organism of industrial importance. We report the development of a gene-knockdown system in this organism by engineering a type I-E clustered regularly interspaced short palindromic repeat (CRISPR)-Cascade system. We used this system to silence an assayable acid phosphatase gene, phoN to 10% of its activity. The study further shows the application of the Cascade system to target an essential gene ssb, that caused poor recovery from radiation. We demonstrate the utility of CRISPR-Cascade to study the role of a regulatory cis-element in radiation response as well as for multi-gene silencing. This easy-to-implement CRISPR interference system would provide an effective tool for better understanding of complex phenomena such as radiation response in D. radiodurans and may also enhance the potential of this microbe for industrial application.

Overview of the Cellular Stress Responses Involved in Fatty Acid Overproduction in E. coli.

Research on microbial fatty acid metabolism started in the late 1960s, and till date, various developments have aided in elucidating the fatty acid metabolism in great depth. Over the years, synthesis of microbial fatty acid has drawn industrial attention due to its diverse applications. However, fatty acid overproduction imparts various stresses on its metabolic pathways causing a bottleneck to further increase the fatty acid yields. Numerous strategies to increase fatty acid titres in Escherichia coli by pathway modulation have already been published, but the stress generated during fatty acid overproduction is relatively less studied. Stresses like pH, osmolarity and oxidative stress, not only lower fatty acid titres, but also alter the cell membrane composition, protein expression and membrane fluidity. This review discusses an overview of fatty acid synthesis pathway and presents a panoramic view of various stresses caused due to fatty acid overproduction in E. coli. It also addresses how certain stresses like high temperature and nitrogen limitation can boost fatty acid production. This review paper also highlights the interconnections that exist between these stresses.

PprM, a Cold Shock Domain-Containing Protein from Deinococcus radiodurans, Confers Oxidative Stress Tolerance to Escherichia coli.

Escherichia coli is a representative microorganism that is frequently used for industrial biotechnology; thus its cellular robustness should be enhanced for the widespread application of E. coli in biotechnology. Stress response genes from the extremely radioresistant bacterium Deinococcus radiodurans have been used to enhance the stress tolerance of E. coli. In the present study, we introduced the cold shock domain-containing protein PprM from D. radiodurans into E. coli and observed that the tolerance to hydrogen peroxide (H2O2) was significantly increased in recombinant strains (Ec-PprM). The overexpression of PprM in E. coli elevated the expression of some OxyR-dependent genes, which play important roles in oxidative stress tolerance. Particularly, mntH (manganese transporter) was activated by 9-fold in Ec-PprM, even in the absence of H2O2 stress, which induced a more than 2-fold increase in the Mn/Fe ratio compared with wild type. The reduced production of highly reactive hydroxyl radicals (·OH) and low protein carbonylation levels (a marker of oxidative damage) in Ec-PprM indicate that the increase in the Mn/Fe ratio contributes to the protection of cells from H2O2 stress. PprM also conferred H2O2 tolerance to E. coli in the absence of OxyR. We confirmed that the H2O2 tolerance of oxyR mutants reflected the activation of the ycgZ-ymgABC operon, whose expression is activated by H2O2 in an OxyR-independent manner. Thus, the results of the present study showed that PprM could be exploited to improve the robustness of E. coli.

Engineering Synthetic Multistress Tolerance in Escherichia coli by Using a Deinococcal Response Regulator, DR1558.

Cellular robustness is an important trait for industrial microbes, because the microbial strains are exposed to a multitude of different stresses during industrial processes, such as fermentation. Thus, engineering robustness in an organism in order to push the strains toward maximizing yield has become a significant topic of research. We introduced the deinococcal response regulator DR1558 into Escherichia coli (strain Ec-1558), thereby conferring tolerance to hydrogen peroxide (H2O2). The reactive oxygen species (ROS) level in strain Ec-1558 was reduced due to the increased KatE catalase activity. Among four regulators of the oxidative-stress response, OxyR, RpoS, SoxS, and Fur, we found that the expression of rpoS increased in Ec-1558, and we confirmed this increase by Western blot analysis. Electrophoretic mobility shift assays showed that DR1558 bound to the rpoS promoter. Because the alternative sigma factor RpoS regulates various stress resistance-related genes, we performed stress survival analysis using an rpoS mutant strain. Ec-1558 was able to tolerate a low pH, a high temperature, and high NaCl concentrations in addition to H2O2, and the multistress tolerance phenotype disappeared in the absence of rpoS. Microarray analysis clearly showed that a variety of stress-responsive genes that are directly or indirectly controlled by RpoS were upregulated in strain Ec-1558. These findings, taken together, indicate that the multistress tolerance conferred by DR1558 is likely routed through RpoS. In the present study, we propose a novel strategy of employing an exogenous response regulator from polyextremophiles for strain improvement.

Expression and mutational analysis of DinB-like protein DR0053 in Deinococcus radiodurans.

In order to understand the mechanism governing radiation resistance in Deinococcus radiodurans, current efforts are aimed at identifying potential candidates from a large repertoire of unique Deinococcal genes and protein families. DR0053 belongs to the DinB/YfiT protein family, which is an over-represented protein family in D. radiodurans. We observed that dr0053 transcript levels were highly induced in response to gamma radiation (γ-radiation) and mitomycin C (MMC) exposure depending on PprI, RecA and the DrtR/S two-component signal transduction system. Protein profiles demonstrated that DR0053 is a highly induced protein in cultures exposed to 10 kGy γ-radiation. We were able to determine the transcriptional start site of dr0053, which was induced upon irradiation, and to assign the 133-bp promoter region of dr0053 as essential for radiation responsiveness through primer extension and promoter deletion analyses. A dr0053 mutant strain displayed sensitivity to γ-radiation and MMC exposure, but not hydrogen peroxide, suggesting that DR0053 helps cells recover from DNA damage. Bioinformatic analyses revealed that DR0053 is similar to the Bacillus subtilis protein YjoA, which is a substrate of bacterial protein-tyrosine kinases. Taken together, the DNA damage-inducible (din) gene dr0053 may be regulated at the transcriptional and post-translational levels.

Hsp20, a small heat shock protein of Deinococcus radiodurans, confers tolerance to hydrogen peroxide in Escherichia coli.

The present study shows that DR1114 (Hsp20), a small heat shock protein of the radiationresistant bacterium Deinococcus radiodurans, enhances tolerance to hydrogen peroxide (H2O2) stress when expressed in Escherichia coli. A protein profile comparison showed that E. coli cells overexpressing D. radiodurans Hsp20 (EC-pHsp20) activated the redox state proteins, thus maintaining redox homeostasis. The cells also showed increased expression of pseudouridine (psi) synthases, which are important to the stability and proper functioning of structural RNA molecules. We found that the D. radiodurans mutant strain, which lacks a psi synthase (DR0896), was more sensitive to H2O2 stress than wild type. These suggest that an increased expression of proteins involved in the control of redox state homeostasis along with more stable ribosomal function may explain the improved tolerance of EC-pHsp20 to H2O2 stress.

Recombinant D. radiodurans cells for bioremediation of heavy metals from acidic/neutral aqueous wastes.

The stability and superior metal bioremediation ability of genetically engineered Deinococcus radiodurans cells, expressing a non-specific acid phosphatase, PhoN in high radiation environment has already been established. The lyophilized recombinant DrPhoN cells retained PhoN activity and uranium precipitation ability. Such cells also displayed an extended shelf life of 6 months during storage at room temperature and showed surface associated precipitation of uranium as well as other metals like cadmium. Lyophilized cells, immobilized in polyacrylamide gels could be used for uranium bioprecipitation in a flow through system resulting in 70% removal from 1mM input uranium solution and a loading of 1 g uranium/g dry weight cells. Compared with a batch process which achieved a loading of 5.7 g uranium/g biomass, the efficiency of the column process was low due to clogging of the column by the precipitate.

PhoN-expressing, lyophilized, recombinant Deinococcus radiodurans cells for uranium bioprecipitation.

Employment of genetically engineered radiation resistant organisms to recover radionuclides/heavy metals from radioactive wastes is an attractive proposition. Cells of recombinant Deinococcus radiodurans strain expressing, a non-specific acid phosphatase encoding phoN gene, were lyophilized. Lyophilized recombinant Deinococcus cells retained viability and PhoN activity and could efficiently precipitate uranium from aqueous solutions for up to six months of storage at room temperature. Batch process for uranium removal using lyophilized cells was more efficient compared to a flow through system, in terms of percent uranium removed, substrate conservation and time taken. Lyophilized recombinant Deinococcus cells exhibited high loading of up to 5.7 g uranium/g dry weight of cells in a batch process at 20 mM input uranium concentration. Lyophilization deflated the cells but did not alter gross cell morphology or surface nucleation capability of cells for uranium precipitation. The precipitated uranyl phosphate remained tightly associated with the cell surface, thus facilitating easy recovery.

Engineering of Deinococcus radiodurans R1 for bioprecipitation of uranium from dilute nuclear waste.

Genetic engineering of radiation-resistant organisms to recover radionuclides/heavy metals from radioactive wastes is an attractive proposition. We have constructed a Deinococcus radiodurans strain harboring phoN, a gene encoding a nonspecific acid phosphatase, obtained from a local isolate of Salmonella enterica serovar Typhi. The recombinant strain expressed an approximately 27-kDa active PhoN protein and efficiently precipitated over 90% of the uranium from a 0.8 mM uranyl nitrate solution in 6 h. The engineered strain retained uranium bioprecipitation ability even after exposure to 6 kGy of 60Co gamma rays. The PhoN-expressing D. radiodurans offers an effective and eco-friendly in situ approach to biorecovery of uranium from dilute nuclear waste.